Method for assessing genotoxicity of a compound

ABSTRACT

The invention concerns a method for assessing in vitro the genotoxicity of a compound, which consist in contacting said compound with at least a cell or cell type overexpressing bcl2 protooncogene and/or related anti-apoptotic protein, and observing the genotoxic effects of said compound on said cell.

The invention relates to a method for assessing the genotoxicity of acompound liable to represent a risk to humans or animals.

At a time when combinatorial chemistry is developing, enabling thesynthesis of countless novel compounds, short-term toxicological studiesare occupying an increasingly important place in the context of thefirst steps of the development of these compounds, liable to be of valuein industries such as the pharmaceutical, agronomic, food and cosmeticsindustries, etc.

Specifically, the development of a novel compound involves studies whichare essential before the very first human exposure and which constitutethe “prerequisites”. These at least involve studies of toxicity bysingle and subacute administration and basal assessment of the mutagenicand genotoxic potential.

Since most carcinogenic substances are mutagenic, and vice versa, it isessential for genetic toxicology to use reliable tests which make itpossible to come to safe conclusions regarding the potentially mutageniceffects of a compound, by gene mutations, chromosomal mutations orgenomic mutations in somatic or germinal cells.

In order to assess the genotoxicity of the compounds of interest,industry makes use of tests which make it possible to demonstrate aclastogenic effect (breaking of chromosomes) or an aneugenic effect(spindle abnormality).

However, the authors of the present invention have shown that thedemonstration of a clastogenic effect may be disturbed by compoundswhich are not clastogenic but which induce apoptosis with the presenceof nuclear events. These tested compounds therefore emerge as“false-positives” or “exaggerated-positives” and are ruled out of anysubsequent use.

The authors of the present invention have developed a method forassessing the genotoxicity of a compound, which makes it possible to berid of this problem of “false-positives” or “exaggerated-positives”.

More precisely, a subject of the invention is a method for assessing thegenotoxicity of a compound, in vitro, in which said compound is broughtinto contact with at least one cell or cell type overexpressing the bcl2proto-oncogene and/or a related anti-apoptotic protein, and thegenotoxic effects of said compound on said cell are observed.

In other words, a subject of the invention is the use of cellsoverexpressing the bcl2 proto-oncogene and/or a related anti-apoptoticprotein, for assessing the genotoxicity of a compound by being free ofthe effects linked to apoptosis alone.

The compound to be tested may be any compound of natural or syntheticorigin, which is designated indifferently “compound”, “product” or“substance”. It may be a mixture of several molecules, which may or maynot be identified, such as, for example, an extract of animal or plantorigin. The compound to be tested may be of therapeutic value, or may beuseful in the chemical, agrochemical, food or cosmetics industry inparticular.

According to a first embodiment of the invention, these positivegenotoxic effects may be observed by the formation of a micronucleus ormicronuclei.

This micronucleus test, described in particular by Matsuoka et al., 1993and Kirsch-Volders et al., 1997, is based on the following principle:

During mammalian cell mitosis, chromosome fragments or whole chromosomeswhich have not undergone segregation will not be located in the mainnucleus during telophase and may be observed, under the microscope, inthe form of micronuclei separated from the main nucleus.

The DNA fragments which give rise to the micronuclei may be caused byeither lesions to DNA (clastogenic or aneugenic effects of genotoxiccompounds) or cleavage subsequent to apoptosis (effects of pro-apoptoticcompounds).

According to a second embodiment of the invention, positive genotoxiceffects of a compound may be observed by the presence of abnormalitiesof number and/or of structure of the chromosomes in metaphase.

This metaphase analysis test has, in particular, been described by Evanset al., 1987.

The principle of this test is as follows: the cells are treated with thecompound to be tested, and then, by taking out and staining the cellsblocked in metaphase (with a blocking agent such as colcemid),chromosomal abnormalities (breaks, rearrangements, etc.) are sought.

These two embodiments (micronucleus test and analytical test inmetaphase) may advantageously be carried out in a complementary fashion.

They do not exclude other embodiments of the method of the invention,using cells overexpressing bcl2 and/or a related anti-apoptotic protein.

Moreover, since some compounds require metabolic activation in order toexert their genotoxic effects, it is possible to add, to the compound tobe tested, a metabolic activator or activation system, such as “S9 mix”,containing a subcellular microsomal fraction of rat liver (Kirkland etal., 1989), in the method of the invention.

In accordance with the present invention, the compound to be tested isbrought into contact with cells overexpressing the bcl2 proto-oncogeneand/or a related anti-apoptotic protein. The overexpression of bcl2,known to be an inhibitor of apoptosis, creates an imbalance betweeninducers and inhibitors of apoptosis of the bcl2/bax family. Theoverexpression of the bcl2 protein thus prevents the cells fromundergoing apoptosis.

The expression “bcl2-related anti-apoptotic protein” is intended to meanany protein of the bcl2 family which has anti-apoptotic activity, thecharacteristics of this family being described by Biolo et al., 1999.

The homology between the various proteins of this family is concentratedin three regions, named BH1, BH2 and BH3, which control their abilitiesto dimerize with other members of the same family and also theirapoptosis-regulating functions. All the anti-apoptotic members alsocontain a BH4 domain located close to their N-terminal end. Theseproteins also have, at their C-terminal end, hydrophobic amino acidswhich appear to be important for anchoring them in intracellularmembranes.

Among the bcl2-related anti-apoptotic proteins, mention maypreferentially be made of bcl-XL, which exhibits very strong homologywith bcl2 (Biolo et al., 1999; Chao et al., 1995).

The cells used are eukaryotic cells, and preferentially mammalian cells.According to a preferred embodiment of the invention, they are CTLL-2cells. CTLL-2cells, which are well known to those skilled in the art,originate from a continuous line of cytotoxic T lymphocytes which is asubclone derived from the C57bl/6 mouse. This line is available at theAmerican Type Culture Collection under the number ATCC TIB-214 and hasbeen described in a certain number of publications (Gillis et al., 1997;Hu et al., 1997). Other cell types may also be used, such as, forexample, L5178Y, CHO, V79, fibroblasts or human or animal lymphomacells, or other eukaryotic cells. A CTLL-2 line can be transfected witha plasmid containing bcl2 or a related sequence, according to standardtransfection and transformation methods known to those skilled in theart. The preparing of CTLL-2-bcl2 cells has been described, inparticular, in the article by Deng et al., 1993.

In accordance with the present invention, the cells may be transfectedso as to overexpress a bcl2-related anti-apoptotic protein, such asbcl-XL, according to standard methods within the scope of those skilledin the art who are aware of the sequences of the corresponding genes(Bolse et al., 1993).

The method in accordance with the invention is efficient for detectingfalse-positives due to the apoptotic phenomenon. In addition, the methodmakes it possible to detect products which have a true clastogenic oraneugenic capacity without being an inducer of apoptosis.

Sensitivity and Reproducibility of the present method make it a validmethod for assessing the genotoxicity of compounds on a large scale.

The following figures and examples illustrate the invention withoutlimiting the scope thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

In all the figures, the stars represented have the following meaning:

*: p<0.05 against the negative control

**: p<0.01 against the negative control

FIG. 1A is a graph which represents an analysis of the metaphases ofhuman lymphocytes treated with dexamethasone for 44 hours.

FIG. 1B represents the measurement of apoptosis in the human lymphocytestreated with dexamethasone, according to the Annexin-V-FITC detectionmethod.

FIG. 2A represents an analysis of the metaphases of CTLL-2 cells (solidline) and CTLL-2-bcl2 cells (broken line) treated with dexamethasone.

FIG. 2B represents the measurement of apoptosis in the CTLL-2 cells(hatched columns) and CTLL-2-bcl2 cells (white columns) treated withdexamethasone, according to the Annexin-V-FITC detection method.

FIG. 3A represents the number of micronucleated cells among the CTLL-2cells (solid line) and CTLL-2-bcl2 cells (broken line) treated withdexamethasone.

FIG. 3B represents the measurement of apoptosis in the CTLL-2 cells(hatched columns) and CTLL-2-bcl2 cells (white columns) treated withdexamethasone, according to the Annexin-V-FITC detection method.

FIG. 4A represents the number of micronucleated cells among the humanlymphocytes treated with etoposide for 4 hours.

FIG. 4B represents the measurement of apoptosis in the human lymphocytestreated with etoposide, according to the Annexin-V-FITC detectionmethod.

FIG. 5A represents an analysis of the metaphases of human lymphocytestreated with etoposide for 4 hours.

FIG. 5B represents the measurement of apoptosis in the human lymphocytestreated with etoposide, according to the Annexin-V-FITC detectionmethod.

FIG. 6A represents the number of micronucleated cells among the CTLL-2cells (solid line) and CTLL-2-bcl2 cells (broken line) treated withetoposide.

FIG. 6B represents the measurement of apoptosis in the CTLL-2 cells(hatched columns) and CTLL-2-bcl2 cells (white columns) treated withetoposide, according to the Annexin-V-FITC detection method.

FIG. 7A represents an analysis of the metaphases of the CTLL-2 cells(solid line) and CTLL-2-bcl2 cells (broken line) treated with etoposide.

FIG. 7B represents the measurement of apoptosis in the CTLL-2 cells(hatched columns) and CTLL-2-bcl2 cells (white columns) treated withetoposide, according to the Annexin-V-FITC detection method.

EXAMPLES Example 1 Micronucleus Test in CTLL-2 and CTLL-2-bcl2 Cells

A—Cell Systems

1) CTLL-2 lymphocytic Cells

The CTLL-2 murine line is a subclone of cytotoxic T lymphocytes derivedfrom the C57bl/6 mouse, the proliferation of which is dependent onrecombinant human interleukin-2 (IL-2).

The cells are cultured in a complete RPMI 1640 medium C1 (Gibco BRL),with:

10% of fetal calf serum decomplemented for 30 minutes at 56° C. (GibcoBRL)

0.01% of 20 mg/ml sodium pyruvate (Sigma)

2 mM of L-glutamine (Gibco BRL)

22 mM of HEPES (Sigma)

100 IU/ml of penicillin (Gibco BRL)

0.1 mg/ml of streptomycin (Gibco BRL)

5×10⁻⁵ M of β-mercapto ethanol (Merck)

1 ng/ml of IL-2

The protocols for freezing, thawing and maintaining the cells at 37° C.are as follows:

freezing of CTLL-2 and CTLL-2 Bcl2 cells

The cells are centrifuged for 5 minutes at 200 g and the pellet isadjusted to 3.5×10⁶ cells per 0.5 ml of decomplemented fetal calf serum,and then 0.5 ml of the cell suspension is transferred into a cryotubecontaining 0.4 ml of decomplemented fetal calf serum and 0.1 ml of DMSO(Merck; batch K2308267-651), at 3.5×10⁶ cells/ml.

The cells are gradually frozen (−1° C. per minute) and stored in liquidnitrogen at −196° C.

thawing of CTLL-2 and CTLL-2 Bcl2 cells

The cryotube is placed in a water bath at 37° C.

The content is transferred into 49 ml of complete RPMI 1640 medium C1and then diluted to {fraction (1/10)}th in C1 medium.

After 24 hours in an incubator at 37° C. under an atmosphere at 5% CO₂and 95% humidity, in a 50 ml flask (Falcon, Becton Dickinson), theculture is centrifuged for 5 minutes at 200 g and the pellet isresuspended in 50 ml of C1 and conditioned in 10 ml flasks.

maintaining of CTLL-2 and CTLL-2 Bcl2 cells

The CTLL-2 cells are cultured in C1 medium which is renewed twice aweek. The cells, which are counted in Malassez chambers, are dilutedsuch that, at the following passage, the density does not exceed 300 000cells/ml, given that the doubling time is 12 hours.

The cultures are placed in an incubator at 37° C. under an atmosphere at5% CO₂ and 95% humidity.

2) CTLL-2-bcl2 Cells

The CTLL-2-bcl2 cells are derived from the transfection, byelectroporation, of CTLL-2 cells with the plasmid pSFFV-bcl2-neo(plasmid provided by the laboratory of Dr. Korsmeyer, WashingtonUniversity, Saint Louis, Mo., plasmid reference: 3088) containing a 1.9kb Eco-RI insert encoding the human bcl2 protein placed under thecontrol of the LTR region of the SV40 virus, and also a gene forresistance to ampicillin and to geneticin (G418) (Renvoizé et al.,1997).

The plasmid is linearized with the Scal restriction enzyme, in aproportion of 1 unit per cleavage site per μg of plasmid, at 37° C. for16 hours. The enzyme is then inactivated at 60° C. for 15 minutes andthe plasmid is precipitated in 100% ethanol. The cells are transfectedwith the plasmid (10 μg of plasmid per 10 million cells) byelectroporation (Biorad, 250 V, 960 μF). The cells are then put backinto culture in complete medium for 48 hours, and the stabletransfectants are then selected in the presence of 800 μg/ml ofgeneticin (Gibco) for at least 15 days, and then by IL-2 deprivation for48 hours. The cells thus selected are cloned by dilution in 96-wellplates (Costar), in a proportion of one cell every two wells.

The freezing, thawing and maintaining of the CTLL-2-bcl2 cells arestrictly the same as for the CTLL-2 cells.

3) Peripheral Lymphocytes

The samples of human lymphocytes are obtained by isolation on a ficollgradient (Hypaque 1077; Sigma; batch 077H6024), after two-fold dilutionof the peripheral blood in RPMI 1640 (Gibco BRL; batch 3001360)containing 0.08‰ of heparin (Sanofi) for a better yield.

The blood was collected by venous puncture using the vacutainer systemon lithium heparin (Becton Dickinson), under sterile conditions, from ahealthy volunteer donor who was a nonsmoker, had not received any recentmedical treatment or radiation, and had not been affected by any recentviral infection.

The cells are cultured at 37° C. in a complete RPMI 1640 (Gibco BRL)medium C2, with:

20% of fetal calf serum decomplemented for 30 minutes at 56° C. (GibcoBRL)

22 mM of L-glutamine (Gibco BRL)

100 lU/ml of penicillin (Gibco BRL)

0.1 mg/ml of streptomycin (Gibco BRL)

100 IU of heparin

+2 ml of phytohemagglutinin A (Wellcome) per 100 ml of medium.

B—Products Studied

1) Aneugenic Agents

griseofulvin

Griseofulvin is an inhibitor of microtubule formation. It is anantimitotic agent: it disturbs the mitotic spindle by virtue of itsinteraction with proteins associated with the polymerized microtubules.The griseofulvin (Sigma; batch 85H07391) is dissolved at 200 mM in DMSO(dimethyl sulfoxide), aliquoted and stored at −20° C.

taxol

Taxol is an antineoplastic agent with proven clinical effectiveness.This compound has aneugenic effects and is an apoptosis inducer.

The taxol (Sigman; batch 126H1382) is dissolved at 400 nM in DMSO,aliquoted and stored at −20° C.

nocodazole

Nocodazole affects mictrotubules and therefore causes aneuploidy. Inaddition, like taxol, it causes phosphorylation of bcl2, stopping of thecell cycle in the M phase and apoptosis.

diethylstilbestrol

Diethylstilbestrol (DES) is an estrogen, the pharmacological propertiesof which have been exploited for the prevention of spontaneousabortions, and which continues to be used in humans in prostate cancertherapy. This compound increases or decreases the transcription of genesregulated by hormones.

It has been reported that DES has carcinogenic actions in humans, inparticular after administration during pregnancy, by inducing aneuploidysubsequent to its effect an centromeres and centrioles.

DES also proves to have clastogenic activity after metabolic activation.

diazepam

Like DES, diazepam is an aneugenic agent. This compound is part of thebenzodiazepines and has an anxiolytic and anticonvulsive effect.

2) Clastogenic Agents

mitomycin C

Mitomycin C is an antibiotic which engenders single-strand cleavages ofDNA and breaking of chromosomes. This compound is a possible carcinogenin humans.

Mitomycin C (Ametycine; batch 440) is dissolved at 500 μg/ml in waterfor injectable preparations, aliquoted and stored at −20° C.

methyl methanesulfonate

Methyl methanesulfonate (MMS) is a monofunctional alkylating agent whichacts by destabilizing DNA, leading to the breaking thereof. It is apossible carcinogen in humans.

The MMS (Aldrich; batch 030177) is dissolved at 7 mM in the RPMIaliquoted and stored at −20° C.

etoposide

The planar cyclic central region, flanked by a phenyl group and by asugar, makes etoposide a nonintercalating, specific and Sphase-dependent inhibitor of topoisomerase II.

This ubiquitous enzyme controls the topology of DNA via the relaxing ofsupercoiled DNA and the catenation and decatenation thereof duringreplication, transcription and cell division. This enzyme is essentialfor chromosome segregation, recombination and separation of chromatids.Etoposide blocks the catalytic activity of topoisomerase 11 bystabilizing the DNA-topoisomerase II complex. Thus, this inhibitionproduces DNA strand cleavages, with sister chromatid exchanges,chromosomal aberrations and chromosome number abnormalites.

It is an apoptosis inducer: etoposide creates breaks in the geneticmaterial and the P53 protein then stops the cell cycle in the G1 phaseso as to allow the transcription of DNA repair genes. When the number ofbreaks is too high, this exceeds the potential of the repair complexesto come to the aid of the genetic material. P53 then intervenes totrigger the programme of cell death. P53-dependent apoptosis isobserved.

These properties explain the fact that etoposide is both a clastogenicagent and an apoptosis inducer.

Etoposide (Sigma; batch 57H1159) is dissolved at 20 mM in DMSO,aliquoted and stored at −20° C.

MNNG and MNU

N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea(MNU) are two alkylating agents which induce DNA lesions of theO6-methylguanine type, which are known to be mutagenic lesions. Thesecompounds produce lethal lesions which are repaired by a mechanism otherthan that involving alkyltransferase.

genistein

Genistein is an isoflavone which is abundant in soya-derived products.It behaves like both an agonist and an antagonist of estrogen receptors.It also has the effect of inhibiting protein tyrosin kinases (PTKs) andtopoisomerases II, and of inducing cellular differentiation andoxidation events. The genistein comes from Sigma and is stored in DMSOat −20° C.

cyclophosohamide

Cyclophosphamide (CPA) is metabolized to a DNA-alkylating intermediate,the effect of which is interference with DNA synthesis and cell divisionin a phase-independent manner. It is clearly established that, in normalcells of the bone marrow or of the intestinal epithelium, blockingoccurs in the G1/S phase so as to repair the lesions or to enter intoapoptosis.

3) Apoptosis Inducers

dexamethasone

Dexamethasone is a glucocorticoid which induces apoptosis according totwo models; the transcriptional model requires activation of cell deathgenes via the glucocorticoid receptor, and therefore the molecule doesnot act on the DNA. Apoptosis may occur according to a model oftransrepression, in which factors required for cell survival would berepressed.

The dexamethasone (Sigma; batch 116H0427) is dissolved at 150 mM inDMSO, aliquoted and stored at −20° C.

gliotoxin

Gliotoxin is a fungal metabolite which induces apoptosis by creatingprotein kinase A-dependent hyperphosphorylation on the serine residuesof the H3 histones, making the cells sensitive to the effects ofnucleases. The gliotoxin comes from Sigma and is stored in DMSO at −20°C.

methional

Methional, which is a metabolite of methionine, is an apoptosis-inducingagent.

C1—Protocol for the Micronucleus Test Without Metabolic Activation

The compounds tested are as follows: griseofluvin, taxol, mitomycin C,MMS, etoposide, dexamethasone, genistein, gliotoxin, methional,nocodazole and DES.

Implementation of the test on CTLL-2 and CTLL-2 Bcl2 cells

2×10⁶ cells are added to 15 ml tubes, each containing 5 ml of completeRPMI 1640 medium supplemented with 25 μg/ml of IL-2. The product to bestudied is then added at various concentrations prepared on a 2-foldbasis. In parallel, a tube is treated with the solvent of the product tobe studied (in the case of DMSO, a final concentration of 0.2% will notbe exceeded) and another is treated with a positive reference product(Mitomycin C at 75 ng/ml; Boehringer; batch 1397592137) in aqueoussolution. The tubes are screwed shut and gently vortexed, and thenplaced in an inclined position in an incubator at 37° C. withoutagitation.

The CTLL-2 cells, which originate from a permanently dividing continuousline, are all targets for the agent studied, and it is therefore notessential to treat them with cytochalasin B (the effect of which is toblock cytokinesis). It is not necessary to limit the analysis tobinucleated cells. In addition, cytochalasin B is likely to interferewith the clastogenic potential of the products of interest.

The lymphocytes are harvested at the 15th hour after the start of theincubation (1.5 cell cycles). The cells are washed twice with 5 ml ofRPMI 1640 supplemented with 2% of decomplemented fetal calf serum. Thecells are then harvested by centrifugation for 5 minutes at 200 g, andthen subjected to a hypotonic shock for 8 minutes (1 volume of RPMI1640:4 volumes of water for injectable preparations +2% ofdecomplemented fetal calf serum). After centrifugation, as muchsupernatant as possible is removed and the cells are fixed with 10 ml ofCarnoy's fixative mixture II (3 volumes ethanol:1 volume acetic acid)for 10 minutes.

After a further centrifugation, the cells are plated out onto slides andleft to dry for 24 hours in the open air, and then stained for 10minutes with Giemsa reagent diluted to 5% in water.

The cells are then examined microscopically at 1250× magnification andthe micronuclei present in the mononucleated cells are sought.

Implementation of the test on peripheral lymphocytes

In the case of lymphocytes, division is induced with phytohemagglutininA. Given that only the T lymphocytes divide, it is essential to addcytochalasine B in order to examine only the binucleated cells whichhave undergone mitosis, when searching for micronuclei.

Cells which have undergone 20 hours of preculturing in C2 completemedium supplemented with phytohemagglutinin A (Murex Biotech; batchF067610) are brought into the presence of the product to be studied.

At the 44th hour after the start of the incubation, the circulatinglymphocytes are washed twice with culture medium containing 10% of dfcsin order to remove the product, and then complete medium containingcytochalasin B (Sigma; batch 87H4054) is added, at a final concentrationof 6 μg/ml.

At the 68th hour after the start of the incubation, the lymphocytes areharvested and undergo the same treatment as above. On the other hand,the micronuclei are sought only in the binucleated cells.

C2—Protocol for the Micronucleus Test With Metabolic Activation

Some products require metabolic activation in order to exert theirgenotoxic effects. In vitro, this activation is carried out using the S9Mix. S9 is a subcellular microsomal fraction of liver from rats inducedwith Aroclor. The composition of the S9 mix (Kirkland et al., 1989) isas follows:

2 ml S9 (prepared in the laboratory)

1 ml KCl at 150 g/l

1 ml glucose-6-phosphate at 180 g/l

1 ml NADP at 25 g/l

This assay is intended to determine whether there is a response, interms of genotoxicity or of apoptosis, subsequent to the metabolicactivation of a compound by the S9 Mix.

0.5×10⁶ cells are added to 15 ml tubes, each containing complete RPMI1640 medium supplemented with interleukin 2 at a final concentration of25 μg/ml. The final volume is 5 ml.

0.25 ml of S9 Mix and the product to be tested are added. In thisexample, this product is cyclophosphamide (CPA).

In parallel, a tube is treated with the solvent of the product.

Incubation is then carried out in a water bath at 37° C. for 3 hourswith agitation at low speed (B.M. GYROTORY G76: set at 2.5, i.e.approximately 80 cycles per minute).

At the end of this period of time, the mixture is centrifuged and thesupernatant is removed.

Washing is then carried out twice with 5 ml of RPMI containing 10% ofdecomplemented fetal calf serum.

The mixture is centrifuged and the supernatant is removed down toapproximately the 0.5 ml mark, and 4.5 ml of complete RPMI mediumsupplemented with 25 μg/ml of interleukin 2 are added.

The mixture is returned to the incubator for a further 15 hours.

Harvesting is carried out according to the same procedure as in themicronucleus test without metabolic activation.

D—Measurement of Apoptosis

1) Detection of Apoptosis With Annexin V-FITC (Euromedex)

The cells treated in parallel to the mutagenesis techniques are washedin culture medium in order to remove the product and are suspended inHEPES/NaOH buffer (10 mM HEPES; pH 7.4; 140 mM NaCl; 5 mM KCl; 5 mMCaCl₂) at 10⁶ cells/ml.

100 μl of this suspension are brought into the presence of 90 μl ofAnnexin V-FITC (diluted at 10 μg/ml in HEPES buffer) for 15 minutes inthe dark at laboratory temperature. Finally, 10 μl of propidium iodidediluted in HEPES buffer, at the concentration of 50 μg/ml, are added.

Flow cytometry analysis is performed on Epics Profile (CoulterCoultronic, Margency): the level of fluorescence emitted by theAnnexin-V-labeled cells is determined after it has passed through afilter at 525±10 nm and displayed after logarithmic amplification. Thefluorescence emitted by the propidium iodide is measured at 600 nm.

2) Detection of apoptosis with YOPRO-1 (Molecular Probes, Eugene, Oreg.)The cells treated in parallel to the mutagenesis techniques are washedin culture medium in order to remove the product.

The diluted YOPRO-1 (1V YOPRO-1:100V EPPI) is added in a proportion of 1μl/0.5×10⁶ cells suspended in 0.5 ml of RPMI 1640.

Flow cytometry analysis is performed on Epics Profile (CoulterCoultronic, Margency): the level of fluorescence emitted by theYOPRO-1-labeled cells is determined after it has passed through a 525±10nm filter and displayed after logarithmic amplification.

E—Expression of the Results

The results obtained at the end of the search for micronuclei and of theinduction of apoptosis are studied using the χ² test, by statisticalcomparison of the results with those obtained with the solvent control,this being for the preliminary studies.

For the search for micronuclei in triplicate and the study of apoptosisin duplicate, the authors of the invention performed a single-factoranalysis of variance. In the event of significance, the Dunnett's testfor multiple comparison was performed.

Using analysis of variance for regression, the authors of the inventionverified that a significant correlation exists between the number ofmicronuclei observed or the percentage of apoptotic cells and theconcentration of product.

F—Results

1) Overexpression of the bcl2 Protein Eliminates the False-positives ByApoptosis

The authors of the invention have shown that a true correlation existsbetween the results obtained on the CTLL-2 and CTLL-2-bcl2 murine linesin terms of induction of micronuclei in the case of cells treated with astrictly aneugenic agent (griseofulvin) or clastogenic agents notrecognized as being apoptosis inducers (mitomycin C and MMS). Whateverthe line, the apoptotic phenomenon is not triggered, whereas themicronucleated cell frequencies increase significantly with the dose. Inaddition, the amplitude of the clastogenic and aneugenic manifestationsis comparable in the two lines.

Moreover, in order to prove that the apoptotic effect is added to thegenotoxic effect, the lines were treated with aneugenic or clastogenicagents which are also apoptosis inducers: taxol and etoposide,respectively. It is effectively shown that the CTLL-2-bcl2 model makesit possible to distinguish between the effects due to apoptosis andthose due to clastogenesis or to aneugenesis. Since the cellstransfected with the gene encoding the bcl2 protein have the property ofnot being sensitive to apoptotic agents due to the imbalanceartificially created between the members of the bcl2 family, the highconcentration of the protein is always greater than that of theapoptosis-inducing proteins.

Although they act via different mechanisms of apoptosis induction, thetwo agents engender comparable phenomena: both induce apoptosis and alsothe formation of micronuclei in the CTLL-2 cells. On the other hand, inthe CTLL-2-bcl2cells, there is much less formation of micronuclei. Thedifference in evolution of the number of micronuclei between the CTLL-2line and the CTLL-2-bcl2 line already appears at the first concentrationof taxol (25 nM). Similarly, it occurs at the first dose of etoposide(62.5 nM; FIGS. 6A and 6B).

On the other hand, the line transfected with the gene encoding theapoptosis-inhibiting bcl2 protein reflects the genotoxic effect of theagents studied by eliminating the apoptotic component. Thus, observationof the evolution of the number of micronuclei in FIG. 6A, which showsdifferent slopes in the CTLL-2 and CTLL-2-bcl2 lines, shows that part ofthe induction of the micronuclei by etoposide is due to the apoptoticphenomenon and that part is due to the clastogenic effect per se of thecompound.

The authors of the invention induced the formation of micronuclei andapoptosis only in the CTLL-2 cells at doses between 12.5 nM and 100 nMof dexamethasone (FIGS. 3A and 3B).

In this model, the percentage of micronucleated cells, which was 3‰ inthe negative control, increased to give a maximum of 60‰ from 50 nM ofdexamethasone.

Such results would make it possible to conclude that the dexamethasonehad mutagenic properties, but the number of cells with micronucleiincreased in similar fashion to the increase in the percentage ofapoptotic cells. The latter increased from 6% (in the negative control)to 30.5% according to the methods with Annexin-V-FITC and YOPRO-1, and amaximum was also observed from 50 nM of dexamethasone.

Whatever the dexamethasone concentrations, it was not possible toobserve either an increase in apoptotic cells or an increase inmicronucleated cells in the CTLL-2-bcl2 strain, again suggesting thatthe apoptosis is responsible for the false-positive results.

The studies on dexamethasone, an acknowledged inducer of apoptosis inlymphocytic cells, therefore make it possible to demonstrate the valueof the CTLL-2-bcl2 model in distinguishing a genotoxic product from anapoptotic product. It does not allow any induction of apoptosis toappear, nor any formation of micronuclei, whereas the nontransfectedCTLL-2 line underwent the apoptotic phenomenon, which is accompanied byan increase in aberrant cell rate. By the same token, this shows thatthe source of the micronuclei is the DNA fragments formed byendonucleases activated during the process of apoptosis. Theseapoptosis-derived fragments can be revealed by a characteristic “ladder”image after agarose gel electrophoresis.

The CTLL-2/CTLL-2-bc2 Model Detects Genotoxic Products

In the in vitro micronucleus test, a product is classified as genotoxicif it causes a statistically significant and dose-dependent increase inthe frequency of micronuclei, relative to the negative control.

At the end of the treatments with the purely clastogenic and purelyaneugenic agents (mitomycin C, MMS, griseofulvin), it was possible tonote, in each of the two cell lines, not only a statisticallysignificant increase in the frequency of aberrations, but also adose-dependent increase: the criteria for classifying a product in thegenotoxic product category are therefore satisfied. In addition, the twolines have the same sensitivity to the products with which they werebrought into contact since, for a given concentration of a producttested, the number of micronucleated cells is comparable in the twolines, without there being any visible apoptosis.

The criteria for positive results are also satisfied in the study of theproducts which are clastogenic or aneugenic and, at the same time,apoptosis inducers (etoposide and taxol), in the CTLL-2 and CTLL-2-bcl2lines. However, the latter line also has the property of only revealingthe aneugenic and clastogenic role of these two products. Specifically,with regard to etoposide (clastogenic) and taxol (aneugenic), anincrease in the incidence of micronuclei is noted outside any apoptoticevent. In the case of etoposide, an increase in the frequency ofmicronuclei in the CTLL-2-bcl2 cells, which do not enter into apoptosis,is observed from 12.5 to 100 nM. With taxol, an increase is observed inthe number of micronuclei at 25 nM on CTLL-2 cells and at 50 nM onCTLL-2-bcl2 cells, whereas these doses do not cause apoptosis. Theseobservations show that the CTLL-2-bcl2 line is a good model not only fordetecting false-positives due to apoptosis, but that it also allows acompound to be attributed its genotoxic role.

In addition, studying a pure apoptosis inducer such as dexamethasone,makes it is possible to demonstrate the role of apoptosis as a source offalse-positives, since its effects on the CTLL-2 line are characteristicof a genotoxic product. However, such a potential can in no way beattributed to the role of apoptosis in the line transfected with thegene encoding the apoptosis inhibitor bcl2.

The CTLL-2/CTLL-2-bcl2 Lines Exhibit Good Sensitivity With Respect tothe Products Studied

The quality of the model developed is confirmed, moreover, by the goodsensitivity thereof. It is noted that, for each of the products tested,the responses are comparable between the assays carried out intriplicate and those carried out in screening and those noted with humanlymphocytes in culture (cf. FIGS. 4A and 4B on human lymphocytes incomparison with FIGS. 6A and 6B on CTLL-2 and CTLL-2-bcl2). Moreover, itis observed that the results obtained with the true genotoxic agents(mitomycin C, MMS, griseofulvine) are the same in the two lines, whichmeans that the overexpression of bcl2 does not modify the sensitivity ofthe cells to the aneugenic or clastogenic effect of a product.

Finally, the reproducibility between results obtained in screening andresults obtained in the assays in triplicate, and the repeatability ofthe assays in triplicate, with low standard deviations, make it possibleto conclude that the interpretation of the results is of good quality.

By studying gliotoxin or methional, the authors of the invention were,as in the case of dexamethasone, able to demonstrate the fact that theCTLL-2/CTLL-2-bcl2 model makes it possible to conclude that the productsinducing only apoptosis were responsible for the formation ofmicronuclei, leading to a falsely positive conclusion in terms ofgenotoxicity.

As in the case of the study with dexamethasone, in the CTLL-2 cellstreated with methional at doses ranging from 60.8 μM to 150 μM,induction of apoptosis and formation of micronuclei was observed withinthe same period of time. The level of apoptotic cells reached 24% at thehighest dose, against 6.5% in the control, whereas the number ofmicronucleated cells increased to 98‰, against 11‰ in the control. Inthe determination of micronucleated cells, as in the induction ofapoptosis, the results were statistically significant (p=0.01) from thefirst dose of methional (60.8 μM). Whatever the methional concentration,neither apoptosis nor micronuclei appeared in the cells transfected withbcl2. In this case also, the micronuclei induced in the CTLL-2 cellswere due to apoptosis.

Gliotoxin was also studied at doses ranging from 25 nM to 200 nM in themodel developed. While apoptosis was maintained at a lower rate in theCTLL-2-bcl2 cells, as was the number of micronucleated cells in thenontransfected cells, the level of apoptosis increased from 7.5% to 45%at the highest dose and the number of aberrant cells increased from 5‰to 20‰. In the micronucleus test and in the apoptosis test, the resultsare different from the control (p=0.01) from 100 nM.

Additional results

Nocodazole, genistein, camptothecin, brefeldin A, anisomycin C,curcumin, quinacrin and thapsigargine induce apoptosis via differentmechanisms in the CTLL-2 line.

As in the case of etoposide, an exaggerated response in terms ofmutagenesis is observed in the CTLL-2 line by the appearance ofapoptosis at the high concentrations of products, whereas the magnitudeof the number of micronucleated cells is less in the CTLL-2-bcl2 line.

The CTLL-2/CTLL-2 Bcl2 model makes it possible to differentiate theapoptotic effect from the mutagenic effect when reading the results ofthe micronucleus test on CTLL-2.

MNU, diazepam and ethyl methanesulfonate (EMS) cause a positive responsein the micronucleus test on CTLL-2 and CTLL-2-bcl2, with the sameamplitude in each of the two lines without inducing apoptosis,confirming the fact that this model makes it possible to detect productshaving genotoxic capacities, whether or not the products are apoptosisinducers.

Actinomycin D and staurosporine, as in the case of dexamethasone, forexample, induce a positive response in terms of clastogenesis in theCTLL-2 cells, correlated with the induction of apoptosis. In theCTLL-2-bcl2 cells, neither apoptosis nor genotoxicity is observed,demonstrating the efficiency of the model in eliminating theinterference due to apoptosis in the in vitro micronucleus test.

Results with metabolic activation:

After metabolic activation, cyclophosphamide (CPA), benzo[a]pyrene and7,12-dimethylbenz[a]anthracene (DMBA) induce the formation ofmicronuclei on the CTLL-2 and CTLL-2-bcl2 cells, and also apoptosis onlyon the CTLL-2 cells, showing that the cells are capable of providing aresponse in terms of mutagenesis and of apoptosis subsequent to thetreatment thereof with a direct mutagen.

Example II Metaphase Analysis Test in CTLL-2 and CTLL-2-bcl2 Cells

A—Materials

The cell systems are the same as in Example I (micronucleus test).

B—Principle

The murine cells are treated with the product to be studied, and then,by plating out and staining the cells blocked in metaphase withcolcemid, abnormalities of chromosomal number and structure are sought.

Annexin-V-FITC is the technique selected to determine the percentage ofcells in apoptosis.

The concentrations selected correspond to the doses used in the in vitromicronucleus test in these cells.

C—Protocol

1×10⁶ cells are added to 15 ml tubes, each containing complete RPMI 1640medium.

The product to be studied is then added at various concentrations, thefinal volume having to be 10 ml.

In parallel, a tube is treated with the solvent of the product (in thecase of DMSO, a final concentration of 0.2% is not exceeded) and twoothers are treated with a reference clastogenic product (MMS at 200 μM)and with a reference apoptosis inducer (dexamethasone at 150 nM).

The tubes are screwed shut and gently vortexed, and then placed in aninclined position in an incubator with agitation at 37° C.

The colcemid is added, at the final concentration of 100 ng/ml, at the13th hour of treatment.

At the 15th hour of treatment, the cells are harvested by centrifugationfor 5 minutes at 200 g and subjected to a hypotonic shock (75 mM KCl)for 5 minutes.

After centrifugation, as much supernatant as possible is removed and thecells are fixed with 10 ml of Carnoy's fixative mixture (3V methanol:1Vacetic acid) for 10 minutes.

After a further centrifugation, the cells are plated out onto slides andleft to dry for 24 hours in the open air, and then stained for 10minutes with Giemsa's reagent diluted to 4% in water.

The slides are examined under a microscope and chromosomal aberrations(breaks, rearrangements, etc.) are sought.

D—Expression of the Results

The results obtained at the end of the metaphase analyses are studiedusing a Student's t test. Although the standard deviations do not makeit possible to demonstrate that the distribution is normal, the test canbe used given the size of the sample. The set of numerical aberrationsand the set of cell numbers, with or without gaps, are treated from astatistical point of view using the χ² test, as is the number ofapoptotic cells.

Firstly, the three recognized purely apoptosis inducers, dexamethasone,gliotoxin and methional, were tested in the metaphase analysis testdeveloped in the CTLL-2 and CTLL-2-bcl2 cells with the aim ofdetermining whether the fragmentation which occurs during the apoptoticphenomenon could give rise to the observation of chromosome breaking inthe metaphase-arrested treated cells.

Then, the question of whether the transfection of the apoptosisinhibitor makes it possible to observe a lesser frequency of aberrationswhen the cells are treated with an agent which is both clastogenic andapoptosis-inducing, such as etoposide, was investigated.

E—Results

In the in vitro metaphase analysis on human lymphocytes, dexamethasone(FIGS. 1A and 1B) may be considered to be a clastogenic agent, althoughthis compound is known to be an apoptosis inducer without beinggenotoxic.

The lymphocytes were treated for 44 hours with a series of doses of 7.5μM to 750 μM of dexamethasone, which induced aberrations in adose-dependent manner. From 250 μM of dexamethasone, apoptosis, whichwas approximately 16% in the control, increased to 40% of the cells. 11%of aberrant cells were observed against 2% in the control.

In order to distinguish the part played by apoptosis in the chromosomeand chromatid breaking, the authors of the invention compared theresults obtained in CTLL-2 cells transfected with the apoptosisinhibitor bcl2 relative to the results obtained on nontransfected CTLL-2cells, in an in vitro metaphase analysis, with a dose of between 9.575nM and 150 nM of dexamethasone (FIGS. 2A and 2B). In parallel, theapoptotic induction was measured. In the CTLL-2 cells, the number ofaberrant cells was statistically different (p=0.05) from the control,from 37.5 nM of dexamethasone. 8% of aberrant cells, against 2% in thecontrol, was in fact observed.

As in the human lymphocytes, the authors of the invention observedchromosome and chromatid breaking but no exchange, and within the sameperiod of time, from 37.5 nM of dexamethasone; the apoptosis, which wasapproximately 7% in the control, increased to 18%, reaching 30% at 150nM. This demonstrates the function of apoptosis in the DNA fragmentationin the metaphase analysis.

The treatment of two cell lines, in metaphase analysis, with 9.375 μM to150 μM of methional reveals structural aberrations, such as breaks orgaps, from only the second dose in the nontransfected cells. Thedifference was statistically significant in comparison with the control(p=0.05), with 13% of cells bearing chromosomal aberrations at 18.75 μM(against 3% in the control).

The metaphase analysis for the cells treated using from 25 to 100 μM ofgliotoxin showed a positive response in the CTLL-2 cells, from 50 μM ofgliotoxin, in terms of clastogenesis (10% of aberrant cells against 1%in the control), but a large increase in apoptosis is also noted (17%against 5% in the controls). At a higher dose, the metaphases could nolonger be analyzed, while the apoptosis reached its maximum. The resultsand differences observed in the cells containing the apoptosisinhibitor, against those which were not transfected, show that theapoptotic phenomenon leads to “false-positive” results in terms ofgenotoxicity, as in the micronucleus test.

The in vitro metaphase analysis of the human lymphocytes treated withetoposide reveals a statistically significant increase in the number ofaberrant cells, from 125 nM to 500 nM of the compound, with manychromosomal exchanges and complex rearrangements, which is proof of anattempt at DNA repair.

Beyond this concentration, it is not possible to interpret themetaphases because there are too many breaks and rearrangements, andapoptosis appears from 2 000 nM of etoposide, to reach 47% (whereas itwas approximately 19% in the control).

This test makes it possible to describe etoposide as being a genotoxiccompound (FIGS. 5A and 5B).

In the micronucleus test, the authors of the invention obtained, withhigh concentrations of etoposide, respectively 4.5‰ (against 2.5‰ in thecontrol) and 29‰ (against 5‰ in the control) of micronucleated cellswith complete inhibition of apoptosis. This observation is also valid inthe metaphase analysis for CTLL-2 cells treated with 31.25 to 500 nM ofetoposide (FIGS. 7A and 7B). The experiment reveals a statisticallysignificant increase (p=0.01) in the number of aberrant cells from 62.5nM to 500 nM of the compound, with chromosomal exchanges and complexrearrangements, which is proof of an attempt at DNA repair, and theapoptosis also increased significantly from the first dose of etoposide.On the other hand, in the CTLL-2-bcl2 cells, the difference (p=0.05)with the control appeared from 125 nM of the compound, with noapoptosis.

F—Conclusion

As in the micronucleus test, the latter results make it possible to showthat apoptosis can engender false-positive results in the metaphaseanalysis test in CTLL-2 cells as well, whereas transfection with bcl2eradicates these events.

By virtue of these results, in accordance with those which were observedin the in vitro micronucleus test in the CTL-2/CTLL-2-bcl2 model, a newtest is therefore available: the in vitro metaphase analysis test, whichdetects aberrations of chromosomal or chromatid structure and numericalaberrations.

What is claimed is:
 1. A method for assessing genotoxicity of acompound, in vitro, in which said compound is brought into contact withat least one cell overexpressing a bcl2 proto-oncogene and/or abcl2-related anti-apoptotic protein, and positive genotoxic effects ofsaid compound on said cell are observed as a) formation of amicronucleus or micronuclei or b) presence of abnormalities of numberand/or structure of chromosome in metaphase.
 2. The method as claimed inclaim 1, in which an observation of positive genotoxic effects of thecompound are characterized by a formation of a micronucleus ormicronuclei.
 3. The method as claimed in claim 1, in which positivegenotoxic effects of the compound are characterized by a presence ofabnormalities of number and/or of structure of chromosomes in metaphase.4. The method as claimed in claim 1, in which the cell overexpressedbcl2.
 5. The method as claimed in claim 1, in which the celloverexpressed bcl-XL.
 6. The method as claimed in claim 1, in which thecell is a cell of a CTLL-2 murine line which has been transfected with anucleic acid expressing bcl2 and/or a bcl2-related anti-apoptoticprotein.
 7. The method as claimed in claim 1, in which said compound tobe tested is in the form of a mixture of molecules.
 8. The method asclaimed in claim 1, in which a metobolic activator is added to the cellto the compound to be tested.